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Abstract

An efficient system for plant regeneration of Senna occidentalis from hypocotyl-derived callus was developed. Callus was induced from leaf and hypocotyl explants on MS medium amended with 9.04 μM 2,4-D + 2.22 μM BAP and 10.74 μM NAA + 2.22 μM BAP. Medium browning due to leaching of compounds from callus was encountered and ameliorated through incorporation of 2.84 μM ascorbic acid. Leaf-derived callus showed no shoot induction ability, while hypocotyl-derived callus produced shoots in all cytokinin-amended treatments and also in combination with 2.68 μM NAA. For shoot formation, BAP-augmented treatments were better than medium with Kin added. Rhizogenesis was better on 1/2 MS basal medium with IBA than in the NAA and IAA treatments. Regenerated plants were acclimatized with 94% survival and showed similar morphology to field-grown plants.
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Abstract

To understand the molecular mechanism controlling in vitro plant morphogenesis, a culture system enabling induction of alternative morphogenic pathways (somatic embryogenesis, SE; shoot organogenesis, ORG) in a well defined population of somatic cells is needed. Arabidopsis is the most useful model plant for genomic studies, but a system in which SE or ORG can be induced alternatively in the same type of explant has not been proposed. Immature zygotic embryos (IZEs) of Arabidopsis provide the only explants with embryogenic potential, and have been recommended for studying mechanisms of SE induced in vitro. This study was aimed at defining culture conditions promoting induction of alternative morphogenic pathways: shoot ORG in IZE explants. The established protocol involves pretreatment of IZE explants with liquid auxin-rich callus induction (CIM) medium, followed by subculture on solid cytokinin-rich shoot induction medium (SIM). The method enables efficient shoot induction in Columbia (Col-0) and Wassilewskija (Ws), genotypes commonly used in molecular studies. During 3 weeks of culture up to 90% of Col-0 and 70% of Ws explants regenerated shoots via an indirect morphogenic pathway. We analyzed the qRT-PCR expression patterns of the LEC (LEC1, LEC2 and FUS3) genes, the key regulators of Arabidopsis embryogenesis, in the IZE explants induced to promote shoot ORG. The sharp decline of LEC expression on SIM medium confirmed that culture of Arabidopsis IZE explants enables experimental manipulation of the morphogenic response of somatic cells. A scheme illustrating various in vitro morphogenic responses of IZEs in relation to hormonal treatment is presented.
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